Validation of the traditional medicinal use of a Mexican endemic orchid (Prosthechea karwinskii) through UPLC-ESI-qTOF-MS/MS characterization of its bioactive compounds

Ethnopharmacological relevance The orchid Prosthechea karwinskii is a medicinal orchid in Oaxaca, Mexico, used to treat diabetes, cough, wounds, and burns, prevent miscarriage and assist in labor. Each part of the plant (leaves, pseudobulbs, or flowers) is used by healers for certain treatment conditions, indicating that each part has different biocompounds with specific pharmacological activity. Aim of the study To characterize the biocompounds in extracts from leaves, pseudobulbs, and flowers of P. karwinskii and evaluate their ROS inhibition capacity to associate it with medicinal uses. Materials and methods The compounds present in extracts from leaves, pseudobulbs, and flowers of P. karwinskii were identified by UPLC-ESI-qTOF-MS/MS. The chemical differentiation of each extract was tested by principal component analysis (PCA) using compound intensity values. For each extract, total phenol and flavonoid contents were quantified. Their antioxidant capacity was evaluated ex vivo by inhibition of ROS with DCFH-DA and in vitro with DPPH radical. Results Based on the PCA, it was observed that some compounds were completely separated from others according to the correlation that they presented. The compounds common to all three plant parts were quinic, malic, succinic, azelaic, and pinellic acids. Among the compounds identified, two were exclusive to leaves, four to pseudobulbs, and ten to flowers. Some of the identified compounds have well-known antioxidant activity. The leaves had the highest content of total phenols and flavonoids, and the highest in vitro and ex vivo antioxidant capacity. A strong correlation was observed between phenol and flavonoid contents, and antioxidant capacity ex vivo and in vitro. Conclusions It was found that the bioactive compounds and antioxidant capacity of each part of the plant were associated with its traditional medicinal use. A pharmacological potential was also found in P. karwinskii for further biological studies because of the type of compounds it contained.


Introduction
Prosthechea karwinskii (Mart.) J.M.H. Shaw, an orchid endemic to southeastern Mexico (Villaseñor, 2016;Solano et al., 2020), is traditionally used in Oaxaca, Mexico. This species is appreciated and cultivated in home gardens in many villages. During Easter, people make ornaments with flowers for religious purposes, mainly to decorate Catholic churches and altars (Solano et al., 2010). In traditional medicine, different parts of the plant are used by healers for the treatment of certain conditions: leaves for the control of diabetes; pseudobulbs for diabetes, cough, and healing wounds and burns; flowers to prevent miscarriages, help in childbirth, and calm coughs (Cruz-García et al., 2014).
The health conditions for which P. karwinskii is used in the traditional medicine of Oaxaca are related to the inflammatory processes. Diabetes is characterized by low-grade inflammation with increased proinflammatory markers (Xu et al., 2021). Cough is an inflammation of the airway (Kamimura et al., 2010). Inflammatory cells are involved in the healing process . Miscarriage is associated with an inappropriate inflammatory response in pregnant women (Christiansen et al., 2006;Gao et al., 2020). In labor, there is an inflammatory overload in feto-maternal tissues (Hadley et al., 2018).
Previous studies have shown that separate extracts from the leaves, pseudobulbs, and flowers of P. karwinskii inhibit in vitro coagulation ( Barrag an-Zarate et al., 2016); as well as reduce glucose, triglyceride, total cholesterol, and adipose tissue in Wistar rats with metabolic syndrome (MS) induced (Rojas-Olivos et al., 2017). The extract from the leaves of this species has anti-inflammatory, gastroprotective, and inhibitory effects on reactive oxygen species (ROS) (Barrag an-Z arate et al., 2020). In addition, foliar extract reduces insulin resistance, proinflammatory status, and cardiovascular risk in Wistar rats with MS-induced (Barrag an-Z arate et al., 2021).
Oxidative stress can affect several physiological functions and lead to chronic diseases (Wolfe and Liu, 2007;Shen et al., 2019). ROS are a normal product of metabolism; however, when their concentration exceeds the levels that can be neutralized by endogenous antioxidant systems, oxidative stress occurs. Cell-based assays are an effective way to evaluate ROS inhibition in an ex vivo model. These assays quantify the bioactivity of compounds considering their adsorption, distribution, metabolism, excretion, and bioavailability (Wolfe and Liu, 2007), which ensures their bioefficacy and bioactivity (Thiengkaew et al., 2021). In contrast to chemical assays, cell-based assays reflect how the enzymes of endogenous antioxidant systems act to attenuate oxidative stress (Xu et al., 2021), so they are biologically representative of antioxidant activity (Wolfe et al., 2008;Wolfe and Liu, 2007;Shen et al., 2019). The ability of a plant to inhibit ROS (and thus oxidative stress) could be related to its therapeutic effects since oxidative stress is involved in the pathogenesis of many diseases. Therefore, a decrease in oxidative stress could be a mechanism by which the plant exerts its therapeutic effect.
Phytochemical screening of leaves, pseudobulbs, and flowers extracts from P. karwinskii revealed the presence of several groups of flavonoid families, phenols, anthraquinones, and saponins ( Barrag an-Zarate et al., 2016). Only the compounds present in leaves have been identified in extracts obtained with ultrasound assistance (Barrag an-Z arate et al., 2020). However, it is unknown whether the pseudobulbs and flowers of this species have a different or similar composition to that of its leaves. However, the therapeutics used for P. karwinskii in traditional medicine mainly consist of the preparation of infusions (Cruz-García et al., 2014). Therefore, it is important to evaluate the extracts obtained by a preparation method similar to traditional medicine.
In traditional medicine, P. karwinskii is used to treat chronic degenerative diseases and other health problems related to inflammatory processes, and healers attribute specific medicinal uses to each part of the plant. This plant represents a potential source for the development of pharmaceuticals for health problems worldwide. However, the leaves are the only plant part studied for their biological activity and identification of their bioactive compounds. Our research group found that leaves have a protective effect on cardiovascular risk and regulate parameters associated with metabolic syndrome (Barrag an-Z arate et al., 2021). Therefore, this study aimed to characterize the bioactive constituents present in the leaves, pseudobulbs, and flowers of P. karwinskii and to evaluate their ROS inhibition capacity to associate them with the medicinal use of each part of plant. We aimed to determine if the particular therapeutic properties of each plant part were due to the specific compounds they contain.

Plant material
The vegetal material of P. karwinskii was rescued from discarded specimens that had been used as decorations during the Easter celebration in Zaachila, Oaxaca. To identify the plant, it was compared with a voucher specimen (Solano 4037) deposited at the OAX Herbarium of the Instituto Polit ecnico Nacional. The specimens were divided into pseudobulbs, leaves, and flowers, and each part was dried, ground, and sieved for compound extraction. This study was performed in accordance with Pridgeon et al. (2005), Soto et al. (2007), Villaseñor (2016), and Solano et al. (2020), in which Prosthechea is recognized as an accepted generic name and includes Euchile. Species considered within Euchile are included in the first category, as is the case for E. karwisnkii (Mart.) Christenson.

Soxhlet extraction
The extraction of compounds was performed using a Soxhlet apparatus following the methodology used by Barrag an-Z arate et al. (2016). Therefore, 180 mL of 50% (v/v) ethanol in a water mixture was used as the solvent, and 10 g of the pulverized sample was added. The extraction was performed at 80 C for 2 h.
The main compounds in each plant part were identified by comparing their exact mass and MS/MS spectra with those reported in the MetaboBase libraries of Bruker and Massbank and those previously reported in scientific articles.

Determination of total phenols and flavonoids content
The total phenol content was determined using the Folin-Denis reagent according to the procedure described by Swain and Hillis (1959). Therefore, the extract of each plant part was prepared at a concentration of 5 mg mL À1 . Gallic acid was used as a reference standard, and the results were expressed as mg gallic acid equivalents per gram of sample (mg GAE g-1).
Total flavonoid content was determined as reported by Chang et al. (2002). Each extract was prepared at a concentration of 5 mg mL À1 . Quercetin was used as a reference standard, and the results were expressed as mg quercetin equivalents per gram of sample (mg QueE g-1).

Inhibition of ROS in an ex vivo model cell-based
To measure the antioxidant capacity of the extracts at the cellular level, the DCFH-DA reagent was used to measure the ROS in the cells. Upon entering the cells and reacting with the ROS, it oxidizes and produces 2,7-dichlorofluorescein (DCF), a fluorescent compound whose intensity is directly proportional to the amount of ROS present in cells (Torres-Rodríguez et al., 2016). Therefore, peripheral blood mononuclear cells (PBMC) were used following the method described by Barrag an-Z arate et al. (2020). In a 96-well plate, 1 Â 10 5 cells were suspended in 100 μL of RPMI medium, and 1.22 μL of DCFH-DA (4.10 mM) was added to each well. Subsequently, 50 μL of each extract was added at a concentration of 1000 μg of extract/mL in 0.1% DMSO. The well to which no extract was added was considered the positive control. The plates were incubated for 30 min at 37 C, with 5% carbon dioxide (CO 2 ) and 95% humidity. Subsequently, 100 μL of PBS was added to each well, centrifuged at 1500 rpm for 5 min at 25 C, and decanted to remove the supernatant. Thereafter, 100 μL of H 2 O 2 (350 μM) dissolved in RPMI medium was added to each well and incubated for 30 min at 37 C, with 5% CO 2 and 95% humidity for 5 h. The results are expressed as the percentage inhibition of ROS relative to the positive control.

Determination of in vitro antioxidant capacity
The in vitro antioxidant capacity of each extract was determined using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging method, as described by Scherer and Godoy (2009). Extracts were prepared at a concentration of 4 mg/mL. The results are expressed as % inhibition relative to the negative control (DPPH with methanol and no extract).

Data processing and statistical analysis
Data for total phenol and flavonoid contents, as well as in vitro and cell antioxidant capacity, are presented as mean AE standard deviation (SD). An analysis of variance (ANOVA), which compares different means, was performed, followed by a Tukey's means comparison test, both implemented in Graph Pad 5.0. Differences were considered statistically significant at p < 0.05. In addition, a correlation test between total phenols, flavonoid content, and antioxidant capacity in vitro (DPPH radical inhibition) and ex vivo (ROS inhibition) was performed using the Pearson correlation coefficient.
The raw data files from UPLC-ESI-qTOF-MS/MS analysis of leaves, pseudobulbs, and flowers of P. karwinskii were collected using Data-Analysis (Bruker) and imported into MetaboScape 3.0 (Bruker) for data processing, including peak extraction. The retention time (TR), m/z, and peak intensity were obtained for each compound. Principal component analysis (PCA) and cluster analysis with unweighted pair group method with arithmetic mean (UPGMA) were applied to the var-covar matrix of intensity values, for which the ordination of the compounds by plant part based on their similarities was evaluated. PCA and cluster analysis were performed using the PAST 4.09 software (Hamer, 2022).

Compounds identified in leaves, pseudobulbs, and flowers extracts of Prosthechea karwinskii
Figures 1, 2, and 3 show the UPLC-ESI-qTOF-MS/MS chromatograms from the leaves, pseudobulbs, and flowers extracts of P. karwinskii, respectively. The corresponding lists of compounds identified are given in Tables 1, 2, and 3. Among the identified compounds, quinic, malic, succinic, azelaic, and pinellic acids were found in all three parts of the plant. Neochlorogenic, chlorogenic, and sebacic acids, as well as rutin and N-undecanoylglycine, were found in both the leaves and pseudobulbs. Leaves and flowers share compounds, such as L-(-)-phenylalanine and guanosine.

Principal component analysis based on UPLC-ESI-qTOF-MS/MS of leaves, pseudobulbs, and flowers of Prosthechea karwinskii
The total ionic current (TIC) chromatograms from UPLC-ESI-qTOF-MS/MS analyses of the leaves, pseudobulbs, and flowers of P. karwinskii are shown in Figure 5a, and the PCA graph is shown in Figure 5b. The differences between the TIC chromatograms of the different parts of the plant can be observed with the naked eye ( Figure 5a). For PCA components 1 and 2 accumulated 100% of the variance (79.867% and 20.133%, respectively). The compounds with the highest contribution to Component 1 were kaempferol-3-O-rutinoside (0.67092), rutin (0.39572), quinic acid (0.33716), embelin (0.22808), and chlorogenic acid (0.20955). In contrast, the compounds with the highest contribution to Component 2 were 2-Methyl-2-propanyl 2,3,4,6tetra-O-acetyl-D-glucopiranoside (0.57137), D-tagatose (0.33499), and azelaic acid (À0.30245). Several compounds aggregated in the central part of the PCA graph because their correlation values were very low. It can be observed that there are also points completely separated from the central part, they correspond to compounds with higher correlation values. Figure 6a shows the contribution of all compounds from the leaves, pseudobulbs, and flowers of P. karwinskii to components 1 and 2 of the PCA; positive and negative scores indicate the type of association with the variance of the components. Figure 6b shows a dendrogram of the similarities among parts of the plant based on their compounds, which was performed to assess whether clusters or differentiations were generated from the compounds. The dendrogram joins pseudobulbs and flowers extracts because of their similar compounds and separates them from the leaves extract.
3.3. Total phenols, flavonoids content, antioxidant capacity, and their correlation coefficients in leaves, pseudobulbs, and flowers extracts of Prosthechea karwinskii Table 4 shows the phenol and flavonoid contents, antioxidant capacity, and correlation coefficients in leaves, pseudobulbs, and flowers extracts of P. karwinskii. The leaves extract had the highest total phenol and flavonoid contents, followed by pseudobulbs and flowers extracts.
From the total content of phenolics and flavonoids quantified, the phenolic compounds identified in P. karwinskii (Tables 1, 2, and 3) were neochlorogenic acid and chlorogenic acid, which were present both in leaves and pseudobulbs, whereas the flavonoids identified were  The antioxidant capacity (ex vivo by inhibition of ROS in cells and in vitro by inhibition of DPPH radicals) of leaves, pseudobulbs, and flowers extracts. Leaves and pseudobulbs extracts showed the highest ROS inhibition in cells, whereas the flowers extract exhibited the lowest. Regarding the inhibition of DPPH radicals, the leaves extract showed the highest activity, followed by the pseudobulbs and flowers extracts. A strong correlation was observed between the flavonoids and total phenol content (0.999) of each plant part. There were strong positive correlations between flavonoids and total phenols with the ability to inhibit DPPH radicals (0.998 and 0.997, respectively), as well as between flavonoids and total phenols with the ability to inhibit ROS (0.823 and 0.821, respectively) ( Table 4).

Compounds present in leaves, pseudobulbs, and flowers of Prosthechea karwinskii and its relationship with the orchid's ethnopharmacological properties
The compounds identified here may be responsible for the medicinal properties of P. karwinskii (Cruz-García et al., 2014). Leaves and pseudobulbs are used to treat diabetes (Cruz-García et al., 2014). The possible compounds responsible for this property, identified in both plant parts, are chlorogenic acid and rutin (Ong et al., 2013;Ma et al., 2015;Ghorbani et al., 2017). Chlorogenic acid improves glucose uptake by skeletal muscles, decreases serum glucose levels (Ong et al., 2013), and decreases insulin resistance (Ong et al., 2013;Ma et al., 2015). Rutin improves the glycemic status in diabetes by decreasing small intestinal carbohydrate absorption, inhibiting tissue gluconeogenesis, increasing tissue glucose uptake, stimulating insulin secretion from β-cells, and protecting islets of Langerhans degeneration (Ghorbani et al., 2017). Other compounds with antidiabetic activity were embelin and phloridzin. Embelin, present only in leaves extract, increases antioxidant enzyme activity and regenerates pancreatic β-cell islets of Langerhans . Phloridzin, which is present only in pseudobulbs extract, reduces blood glucose levels in mice (Kobori et al., 2012). These compounds could confer P. karwinskii its pharmacological properties related to diabetes treatment by acting alone or synergistically.
The medicinal uses of P. karwinskii are also related to the inflammatory processes. Quinic acid with inflammation-related activity was identified in all three extracts. This compound inhibits the nuclear factorkappa light chain of activated β-cell (NF-κβ) and mitogen-activated protein kinase (MAP kinase) signaling pathways (Jang et al., 2017). Compounds found in both leaves and pseudobulbs, such as chlorogenic acid, rutin, and neochlorogenic acid, in addition to inhibiting MAP kinase and NF-κβ signaling, can inhibit nitric oxide (NO) production, decrease the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), and decrease the production of proinflammatory cytokines (Hwang et al., 2014;Wang et al., 2012;Yeh et al., 2014;Kim et al., 2015;Young et al., 2018;Zhao et al., 2020). Embelin, present only in leaves extract, can decrease the production of the proinflammatory cytokines interleukin 1 beta (IL-1β), tumor necrosis factor-α (TNF-α), and interleukin 6 (IL-6) (Kumar et al., 2011;Naik et al., 2013). Guanosine, found in leaves and flowers, decreases the expression of iNOS and proinflammatory cytokines TNF-α and IL-1β (Quincozes-Santos et al., 2014). These compounds, acting alone or synergistically, may be responsible for the ability of the plant to treat inflammation-related disorders.
The remedy to prevent miscarriages and help during labor is an infusion with flowers adding a coin or gold jewel, or in some cases, a  potter wasp (Cruz-García et al., 2014). Gold-derived compounds have shown efficiency in treating inflammatory diseases such as rheumatoid arthritis (Ott, 2009;Cuevas et al., 2012); gold nanoparticles can enhance the anti-inflammatory properties of compounds such as chlorogenic acid through gold reduction by OH À oxidation from the caffeic acid moiety present (Hwang et al., 2015). In addition, it can decrease the expression of proinflammatory markers (Muller et al., 2017). Abscisic acid, which is only found in flowers extract, can increase the expression of genes involved in muscle relaxation of the blood vessel wall (Guri et al., 2010). Because of this property, this compound could be related to the use of flowers to prevent miscarriage and aid in labor. Abscisic acid also has an antinociceptive effect (Mollashahi et al., 2018), which could facilitate labor. Potter wasps sometimes added to the flower's infusion (Cruz--García et al., 2014) may decrease pain, as some compounds present in

Phenols and flavonoids in extracts of Prosthechea karwinskii leaves, pseudobulbs, and flowers
Phenolic compounds and flavonoids are known for their biological properties similar to those of P. karwinskii in traditional medicine, as they may exert a protective effect against diabetes (Varshney et al., 2019;Salau et al., 2020;Dinda et al., 2020;Hussain et al., 2020), have anti-inflammatory activity (Bayat et al., 2021;Liskova et al., 2021) and aid in wound healing (Carvalho et al., 2021). Therefore, these compounds might be responsible for the pharmacological properties of P. karwinskii.
Antioxidant is another activity presented by phenols and flavonoids. They trap ROS and inhibit the action of enzymes or trace elements involved in free radical generation (Banafsheh and Sirous, 2016). According to Pearson's correlation coefficients, the presence of a higher content of phenols and flavonoids in the leaves of P. karwinskii is related to a higher antioxidant capacity in this part of the plant than in the pseudobulbs and flowers.

Antioxidant capacity of Prosthechea karwinskii leaves, pseudobulbs, and flowers
Prosthechea karwinskii extracts have antioxidant capacity both in vitro and ex vivo; therefore, they can treat ailments affecting human health caused by oxidative stress. Antioxidant compounds protect organisms from damage caused by free radicals that induce oxidative stress (Ozsoy et al., 2008). Health problems related to oxidative stress include diabetes (Nowotny et al., 2015;Lima Júnior et al., 2021), inflammation-related problems , and miscarriage (Liang et al., 2020;Agarwal et al., 2012). Traditional medicinal therapies for such problems include the use of this plant.
Extracts of the leaves and pseudobulbs of P. karwinskii were able to inhibit ROS production. This could explain the antidiabetic properties of orchid leaves and pseudobulbs in traditional medicine. This is because increased glucose levels auto-oxidize glucose and increase the production of free radicals and ROS (Lotfi et al., 2020), which increase oxidative stress in β-cells, leading to insulin resistance and decreased secretion of this hormone (Karunakaran and Park, 2013;Sheweita et al., 2020;Lima Júnior et al., 2021). In addition, elevated blood glucose levels increase ROS production and the formation of advanced glycation end products, which are factors involved in the development of diabetes mellitus (Nowotny et al., 2015;Lima Júnior et al., 2021).
The use of the plant to treat inflammation-related conditions could also be due to their ability to inhibit ROS, as oxidative stress can cause inflammation by increasing the generation of proinflammatory mediators, such as TNF-α and iNOS . Previously, the ability of P. karwinskii leaves extract to decrease inflammation and inhibit ROS formation has been corroborated (Barrag an-Z arate et al., 2020).
The ability of P. karwinskii flowers extract to inhibit ROS could be related to the medicinal use of this part of the plant in the prevention of abortion. Oxidative stress is a contributing factor to miscarriage (Liang et al., 2020;Agarwal et al., 2012) and the antioxidant defense system of enzymes, such as glutathione peroxidase (GSH-Px), is decreased in women with this condition (Zachara et al., 2001;Omeljaniuk et al., 2015). Furthermore, the addition of gold to the infusion of flowers could improve their antioxidant properties, decrease oxidative stress, and help prevent miscarriage. This is because gold compounds can inhibit the production of ROS (Cuevas et al., 2012) and gold nanoparticles can enhance the levels of antioxidant enzymes, such as SOD, CAT, GSH-Px, and GSH (Muller et al., 2017).
The antioxidant compounds identified in P. karwinskii are rutin (Yeh et al., 2014), kaempferol-3-O-rutinoside (Wang et al., 2015), embelin , and guanosine (Quincozes-Santos et al., 2014;Courtes et al., 2019). These compounds decrease oxidative stress by increasing the antioxidant activity of SOD, CAT, GSH-Px, and GSH. Azelaic acid, also found in P. karwinskii, reduces ROS generation in neutrophils (Akamatsu et al., 1991). Rutin and azelaic acid are present in both leaves and pseudobulbs, and the plant parts have the highest ROS inhibition ability. Embelin and kaempferol-3-O-rutinoside are present only in the leaves, the plant part with the highest inhibition of ROS and DPPH radicals. Guanosine is present in both the leaves and flowers of plants. In the chromatograms (Figures 1, 2, and 3), it was observed that the leaves extract presented a higher intensity of peaks corresponding to compounds with antioxidant capacity, followed by pseudobulbs and flowers extracts, which agrees with the results of antioxidant capacity, both in cells and in vitro.
close relationship between the biological activities reported for these compounds and the traditional uses attributed to the different parts of the orchid in traditional medicine. The compounds unique to each part of the plant contribute to its medicinal properties, although these properties may also be enhanced by the common compounds they share.
According to the PCA analysis, the compounds with the highest contribution to the variance (kaempferol-3-O-ruthinoside, rutin, quinic acid, embelin, and chlorogenic acid) correspond to those present in the leaves, including some compounds exclusive to this part of the plant.

Author contribution statement
Gabriela Soledad Barrag an-Zarate: Conceived and designed the experiments; Performed the experiments; Analyzed and interpreted the data; Wrote the paper.
Luicita Lagunez-Rivera: Conceived and designed the experiments; Analyzed and interpreted the data; Wrote the paper. Rodolfo Solano: Analyzed and interpreted the data; Wrote the paper. Candy Carranza-Alvarez: Performed the experiments; Contributed reagents, materials, analysis tools or data.
Diego Manuel Hern andez-Benavides: Performed the experiments; Analyzed and interpreted the data.
Gerard Vilarem: Contributed reagents, materials, analysis tools or data.

Data availability statement
Data included in article/supp. material/referenced in article.

Declaration of interest's statement
The authors declare no conflict of interest.

Additional information
No additional information is available for this paper. mg GAE g À1 : mg gallic acid equivalent per g of sample; mg QueE g À1 : mg quercetin equivalent per g of sample; ROS: reactive oxygen species, DPPH: 1,1-diphenyl-2picryl-hydrazyl. Results are shown as the mean AE standard deviation. Superscripts with different letters indicate significant differences (P 0.05).